Homologous recombination mRNAs (RAD21, RAD50 and BARD1) have a potentially poor prognostic role in ERBB2-low bladder cancer patients

Human epidermal growth factor receptor 2 (HER2/ERBB2) factor is known to be implicated in many malignancies and the potential of it as a prognostic biomarker was reported years ago. Molecular subtypes of HER2/ERBB2 negative and positive with distinct clinical outcomes have been identified in recent years; however, it is still under investigation for bladder cancer. This study evaluates the biological and prognostic significance of RAD21, RAD50 and BARD1 (homologous recombination biomarkers) mRNA levels with ERBB2 low and high expression to explore their impact on bladder cancer patient survival and cancer aggressiveness. The expression of ERBB2, RAD21, RAD50 and BARD1 mRNA levels was assessed in The Cancer Genome Atlas (TCGA) bladder cancer dataset along with four validation cohorts. Outcome analysis was evaluated using disease-free survival (DFS) and overall survival (OS). Univariate and multivariate analysis were used to evaluate the relationship between RAD21, RAD50, BARD1 and ERBB2 expression and clinicopathological variables. A significant increase in mRNA expression levels of RAD21, RAD50 and BARD1 was noticed in ERBB2-low patients compared to ERBB2-high patients. This overexpression of the homologous recombination repair transcripts was associated with poor outcome in ERBB2-low tumors, not in ERBB2-high tumors. Furthermore, the combined expression of high RAD21/RAD50, high RAD21/BARD1 or high RAD50/BARD1 were significantly associated with worse DFS and a better outcome for those with low co-expression in the ERBB2-low cohort. High expression of either RAD21/RAD50 or RAD21/BARD1 in ERBB2-low cohort associated with higher chance of metastasis. In addition, gene expression of BARD1 alone or in combination with RAD50 acted as an independent prognostic factor for worst survival. The data presented in this study reveal a connection between RAD21, RAD50, BARD1 and ERBB2 and patient survival. Importantly, it provided novel findings and potential prognostic markers, particularly in ERBB2-low bladder cancer.


Gene-gene interaction network construction and analysis. For gene-gene interaction network
between RAD21, RAD50, BARD1 and ERBB2 we used the GeneMANIA Cytoscape plugin (https:// apps. cytos cape. org/ apps/ genem ania) 59,60 . Interaction network covering; physical interactions, co-expression, co-localization, genetic interactions, pathway and shared protein domains. With max 100 genes interaction and max attributes. Network structure was visualized by Cytoscape (https:// cytos cape. org/) 61 . To further analyze and calculate the topology parameters (Node degrees, Betweenness centrality and Closeness centrality) of the network, NetworkAnalyzer 62 , a plugin in Cytoscape, was applied.
Gene ontology (GO) functional and pathway enrichment analysis. To provide Gene Ontology analysis we used the Database for Annotation, Visualization and Integrated Discovery tool (DAVID; latest version Dec. 2021: https:// david. ncifc rf. gov/ home. jsp). This tool includes biological process, molecular function, cellular component and also Kyoto Encyclopedia of Gene and Genomes (KEGG) pathway analysis 63 . Enrichment analysis was performed with the threshold of p < 0.05. Statistical analyses. Data analysis were performed using JMP Pro 15 (SAS Institute Inc., USA). For the prognostic significance survival curves, Kaplan-Meier method was used with log-rank comparison for significance testing. In the univariate analysis, Chi-square test (χ 2 ) was used to evaluate the relationship between RAD21, RAD50, BARD1 and ERBB2 expression and clinicopathological variables. In multivariate analysis, to emphasize on RAD21, RAD50, BARD1 and ERBB2 interaction, a Cox proportional hazard model was used for the multivariate survival analysis including all potential confounder factors. The proportional hazards assumption was checked, the relationship between log cumulative hazard and a covariate was linear. Where appropriate, two-tailed Student's t-test was performed using GraphPad Prism (version 9.5.0, USA). All differences were considered statistically significant at p < 0.05, p values were two-sided; all confidence intervals were at 95%.

Results
Expression of ERBB2, RAD21, RAD50 and BARD1 in bladder tissues. We initially compared the total expression levels of ERBB2, RAD21, RAD50 and BARD1 mRNA in normal and tumor bladder tissues with bioinformatics analyses using the TCGA database (Cohort one). The cohort consists of 413 patients with MIBC and matched normal samples, the TCGA datasets were previously described 64 . The data revealed a significantly high mRNA expression levels of ERBB2 and RAD21 in tumor tissues compared to normal; median = 6.888 tumor vs. 6.299 normal; p < 0.0001 and median = 6.408 tumor vs. 6.043 normal; p = 0.034, Fig. 1A respectively. RAD50 and BARD1 mRNA levels showed no significant difference between tumors and the respective normal tissues (Fig. 1A). Interestingly, when we sub grouped patients according to ERBB2 status (ERBB2-low and ERBB2-high), we found that RAD21, RAD50 and BARD1 expression levels increased significantly in ERBB2-low patients compared to ERBB2-high patients. Figure 1B, shows RAD21 expression median = 0.02 in ERBB2-low compared to RAD21 median = − 0.07 in ERBB2-high; p < 0.0001. RAD50 expression median = 0.12 in ERBB2-low compared to RAD50 median = − 0.02 in ERBB2-high; p < 0.0001. BARD1 expression median = 0.21 in ERBB2-low com- www.nature.com/scientificreports/ pared to BARD1 median = 0.08 in ERBB2-high; p < 0.0001. We validated this finding with Cohort two from MSK dataset (Fig. 1C). As expected, total RAD21 in ERBB2-low cohort increased significantly to the same in ERBB2high cohort (median = 0.29 vs. median = − 0.34; p < 0.0001). Total RAD50 expression in ERBB2-low patients was higher compared to RAD50 in ERBB2-high cohort (median = 0.20 vs. median = − 0.09; p < 0.0001). The same significant trend was shown with BARD1 expression in different ERBB2 status (median = 0.01 vs. median = − 0.24; p < 0.0001). The second validation dataset (Fig. 1D Fig. S2A,B, respectively). RAD50 transcript level (Cohort one) did not influence survival in the whole cohort (p = 0.085; Fig. 2D) and in ERBB2-high cohort (p = 0.971; Fig. 2F). Though high RAD50 mRNA was significantly associated with poor survival in the ERBB2-low cohort (p = 0.007; Fig. 2E). High RAD50 mRNA expression also Furthermore, investigating the homologous recombination repair transcripts (RAD21, RAD50 and BARD1) with each other revealed that combined expression of high RAD21/high RAD50 significantly associated with worst DFS and better outcome for those with low RAD21/low RAD50 in the ERBB2-low cohort (p = 0.017; Fig. 3B). No significant different in the whole cohort and in the ERBB2-high cohort (Fig. 3A,C). Data also showed a tendency toward poor OS with high RAD21/high RAD50 (5-year OS of 29.5%) and better with low RAD21/low RAD50 (5-year OS of 45.4%) in ERBB2-low patients, but the trend was not significant (Additional file 1: Fig. S4A-C). Similarly, combined high RAD21/high BARD1 associated significantly with worst outcome in the whole cohort and ERBB2-low cohort (p = 0.031, p = 0.005; Fig. 3D,E; respectively). Whereas, no significant association was found in the ERBB2-high patients (Fig. 3F). High RAD21/high BARD1 showed a tendency toward poor OS with 5-year of 29.2% vs. 42.5% with low RAD21/low BARD1 in ERBB2-low cohort (Additional file 1: Fig. S4D-F). Then again, low RAD50/ low BARD1 mRNA expression showed a significantly better DFS compared to other subgroups in the whole cohort and in the ERBB2-low cohort (p = 0.019, p = 0.004; Fig. 3G,H; respectively). The OS was also better with 51.5% 5-year rate vs. 31.1% with high RAD50/high BARD1, though not significant (Additional file 1: Fig. S4G,H). Finally, no significant DFS and OS differences were found in any group among the ERBB2-high cohort ( Fig. 3I and Additional file 1: Fig. S4I).

RAD21, RAD50
and BARD1 mRNA levels and clinicopathological features. To further evaluate the impact of RAD21, RAD50 or BARD1 mRNAs with ERBB2 status on the clinicopathological variables, we www.nature.com/scientificreports/ used the TCGA database (Cohort one). We previously described the ERBB2 distribution of the clinicopathological characteristics of this cohort 64 . Univariate analysis data indicate that in ERBB2-high cohort mRNA expression of RAD21 low was significantly associated with tumor grade (p = 0.011). Also, BARD1 low was significantly associated with tumor grade (p = 0.04) and non-papillary tumor shape (p = 0.037). However, no association was observed in ERBB2-low cohort (Table 1). Table 2 summarizes the association between the co-expression of the homologous recombination repair transcripts with ERBB2 and the clinicopathological features. Analyzing the combined high expression of either RAD21/RAD50 or RAD21/BARD1 in ERBB2-low cohort had a significant association with higher chance of metastasis (p = 0.011). On the other hand, low expression of RAD50/BARD1 in ERBB2-low cohort had a significant association with higher tumor stages (p = 0.013). The high expression of RAD50/BARD1 correlated significantly with papillary tumor shape (p = 0.035) ( Table 2). No significant association with any co-expression was observed in ERBB2-high cohort (Additional file 2: Table S1).
Multivariate analysis of RAD21, RAD50 or BARD1 mRNAs expression alone or in combination was conducted. This was done to investigate whether the expressions are an independent prognostic factor. As shown in Table 3, multivariate analyses of the above factors together with tumor stage were conducted. BARD1 mRNAs expression was an independent prognostic factor for worse DFS in the ERBB2-low cohort (p = 0.047, Hazard ratio 1.812, 95% CI 1.009-3.330), but not in ERBB2-high cohort. Similarly, in the ERBB2-low cohort combination of RAD50/BARD1 mRNA expression was an independent factor for poor DFS (p = 0.008, Hazard ratio 1.378, 95% CI 1.088-1.760). Whereas tumor stage was an independent prognostic factor for poor DFS in ERBB2-high cohort (< 0.001, Hazard ratio 1.295, 95% CI 1.154-1.458).

Gene interaction network of RAD21, RAD50, BARD1 and ERBB2. A gene interaction network
was constructed for the three homologous recombination repair transcripts (RAD21, RAD50 and BARD1) and ERBB2. This was done to identify the most related genes network between our targets. The network was constructed using the GeneMANIA Cytoscape plugin 59 Table S2). Interaction percentages in the network were: 82.19% physical interactions, 8.40% co-expression, 3.78% co-localization, 2.99% genetic interactions,            www.nature.com/scientificreports/ 2.01% pathway and 0.64% shared protein domains. In addition, a network interaction was analyzed illustrating the node degrees, betweenness centrality and closeness centrality with Network analyzer of the 100 top genes, as shown in Table 4.

Functional and pathway enrichment analyses.
The functional enrichment pathways of the RAD21, RAD50, BARD1 and ERBB2 were investigated using DAVID software to identify significant GO categories and KEGG pathways. In the molecular function of GO; majority of genes involved in protein binding along with ERBB2, RAD21 and BARD1. ERBB2 and RAD50 appears in both functions of identical protein binding and ATP binding. Likewise, protein heterodimerization activity showed BARD1 and ERBB2. In the cellular component of GO; most genes are enriched in nucleus along with our four target genes. Cytosol shows ERBB2 and RAD21. RAD21, RAD50 and BARD1 were also found in more functions and pathways involves in; cytoplasm, nucleoplasm, plasma membrane, site of double-strand break and macromolecular complex. In the biological process of GO results were; positive regulation of kinase activity involved both ERBB2 and RAD50. Negative regulation of    www.nature.com/scientificreports/ apoptotic process involved ERBB2 and BARD1. DNA repair, cellular response to DNA damage stimulus, doublestrand break repair, cell division and more are suggested to be regulates indirectly with our targeted genes. In the KEGG pathway enrichment analysis data demonstrated that; homologous recombination, ERBB signaling pathway, cell cycle, PI3K-Akt signaling pathway, microRNAs in cancer and pathways in cancer were associated with RAD21, RAD50 and BARD1 (Fig. 4B and Additional file 2: Table S3).

Discussion
Several studies have highlighted the important role of homologous recombination factors (RAD21, RAD50 and BARD1) in cancer progression, aggressiveness and genomic instability in many cancer types 22,24,28,43,65,66 . Though, these factors remain largely unexplored especially in bladder cancer. We recently highlighted the interplay between ATM (one of the homologous recombination factors) and ERBB2 in bladder cancer patients 64 . In the current study, we observed a significant overexpression of ERBB2 in bladder cancer tissues compared to normal. This overexpression was in agreement with many published studies that showed overexpression of ERBB2 in solid cancers, serving as a prognostic and predictive biomarker especially in breast, gastric, colorectal and bladder cancer [67][68][69][70] . Kiss et al., reported that ERBB2 amplification is not always associated with HER2 overexpression in bladder cancer, and HER2 overexpression was observed without gene amplification. Suggesting that both HER2 protein and ERBB2 gene expressions are regulated by different mechanisms 71 . In this current investigation we examined RAD21, RAD50 or/and BARD1 co-expression with different status of ERBB2 expression and assessed their prognostic and clinical significance in bladder cancer.
Published data reported that RAD21 mRNA amplification correlates with gene copy number in grade 3 luminal, basal and HER2 subtypes of breast cancer. Also, RAD21 protein overexpression correlates strongly with gene amplification 27 . This overexpression was implicated in many cancer types and associate with poor outcomes in patients [22][23][24][25][26] . Similarly, RAD21 mRNA was upregulated in bladder cancer tissues compared to normal tissues, also an increase in mRNA level was detected in late-stage bladder cancer cell lines 28 . In agreement with previous data, our data showed significant upregulation of RAD21 expression in bladder cancer tissues compared to normal. To investigate the relation between RAD21 and ERBB2 expression, we sub-grouped all patients cohorts according to ERBB2 status. Interestingly we found that RAD21 mRNA level increase significantly in patients with low-ERBB2 compared to patients with high-ERBB2. Furthermore, as Yu et al., indicated in their whole cohort RAD21 expression alone did not influence survival significantly on the OS 28 . In this study, we confirmed this in OS and DFS, also in both subgroups of ERBB2 cohorts. Interestingly we found high RAD21 mRNA was linked to poor survival in the ERBB2-low cohort in the main MIBC cohort and confirmed it in both high grade and MIBC validation cohorts. Furthermore, we also found this trend in both the low tumor grade subgroup and the early tumor stage (Ta) subgroup of the NMIBC validation cohort. In contrast, RAD21 low mRNA showed significant association with low tumor grade in ERBB2-high cohort. These findings suggest that additional data maybe required in the future to corroborate statistically the impact that RAD21 plays in specific types of bladder cancer.
RAD50 is one of the key players in homologous recombination repair and telomere maintenance 29 . Literature is reporting that RAD50 high expression associated with aggressive high grade cystadenocarcinomas and low RAD50 linked to better progression free survival 72 . The aggressive phenotype and poor survival associated with high RAD50 expression at protein and transcriptomic levels was also reported in bladder, gastric, colorectal, rectal and ovarian cancers [73][74][75][76] . Hence the RAD50 factor role is yet to be elucidated in different cancer types, in the current study we first assessed the total RAD50 mRNA expression level which was not altered in bladder cancer compared to normal tissues. Interestingly, following the subgrouping of cohort according to ERBB2 status, we found that RAD50 mRNA level increased significantly in patients with low-ERBB2 compared to patients with high-ERBB2. This increase was translated to poor DFS for patients with high RAD50 in the ERBB2-low cohort. This finding was confirmed in the MIBC and the high grade cohorts. Moreover, the same trend was seen in the low grade subgroup and the early tumor stage (Ta) subgroup of the NMIBC cohort. These findings further support our conclusion that RAD50 mRNA level may have a poor prognostic role in ERBB2-Low bladder cancer patients, regardless of the grade or stage distribution of the cohort. Further significant associations between clinicopathological variables and RAD50 at different ERBB2 levels were not seen.
BARD1 is another player in the homologous recombination pathway, it was suggested that this role in DNA repair pathway is through direct interact between BARD1 and BRCA1 40,41 . Variants of BARD1 gene were associated with many solid tumors [44][45][46]77 . Hawsawi et al., recently illustrated that high BARD1 mRNA expression was associated with poor OS, relapse free survival and distant metastasis free survival in breast, ovarian and gastric cancer but not lung cancer 43 . In the current study, BARD1 mRNA did not show any alteration in expression level between bladder cancer tissues and normal. Though, significant upregulation was observed in BARD1 mRNA in patients with low-ERBB2 compared to patients with high-ERBB2 in all study cohorts. Interestingly this high BARD1 mRNA was translated to poorer DFS in the whole cohort and in the ERBB2-low cohort in compared to patients with low BARD1, though no significant was detected when ERBB2 expression was high. Based on our analysis, we have observed an association between high levels of BARD1 mRNA expression and poor survival in the main cohort, as well as the validation MIBC cohort. Additionally, we found a similar trend in the NMIBC subgroups, particularly in the low grade and early tumor stage (Ta-T1) patients. This implies that BARD1 mRNA may be a promising prognostic marker for bladder cancer patients, irrespective of the tumor grade or stage. In contrast, BARD1 low expression was significantly associated with low tumor grade and non-papillary tumor shape in ERBB2-high patients. We also showed that BARD1 mRNAs expression was independent prognostic factor for worse DFS in the ERBB2-low cohort, but not in ERBB2-high cohort. Our observations suggest the potential value of the expression pattern of BARD1 at specific subtypes of bladder cancer.
As we highlighted the role of each homologous recombination factors (RAD21, RAD50 or BARD1) to patients' survival and cancer aggressiveness in bladder cancer, other groups studied these factors in different cancer Multivariate analyses data showed that RAD50/BARD1 mRNA expression was independent prognostic factor for poor DFS in the ERBB2-low patients. Therefore, these homologous recombination potential biomarkers may play roles in predicting metastasis and survival in bladder cancer patients. We next sought to investigate the interaction network between RAD21, RAD50, BARD1 and ERBB2 to provide deeper insight into the molecular mechanisms of these relations through identifying the most related genes network between our targets. Overlapping genes were identified with high physical interactions, co-expression, co-localization, genetic interactions, shared pathway and shared protein domains with RAD21, RAD50, BARD1 and ERBB2. These genes include: BRCA1, SMC3, H2AX, EGFR, SMC1A, PCNA, ATM, RPA2, TP53, RPA3, LMNB1, RPA1, BLM, TERF2, STAG1, MRE11, PRKDC, TERF2IP, STAG2, CDK4, TOPBP1, TERF1, POT1 and more. Centrality measure of this network indicates the importance of these intermediate genes to the interaction between our targets. This was followed with the functional and pathway enrichment analysis which showed majority of the overlapping genes with ERBB2, RAD21 and BARD1 involves in protein binding. ERBB2 and RAD50 factors appear in identical protein binding and ATP binding. Moncalian et al., showed how the motif signature is essential to ATP binding and biological function of RAD50 78 . Tarsounas et al., discussed how BARD1 and BRCA1 heterodimers through its E3 ubiquitin ligase activity, then the ability of this heterodimer to interact with other DNA damage response factors through the homologous repair pathway 40 . Our data suggested the involvement of both BARD1 and ERBB2 along with other overlapping genes in protein heterodimerization activity. We also found that many genes are enriched along with our four target genes in the nucleus, which agrees with other studies emphasizing our target genes functional role in localizing to the nucleus to participate in the DNA repair 25,79,80 . In addition, data illustrated that ERBB2 and RAD50 appear in the positive regulation of kinase activity. Similarly, the enriched results also identified ERBB2 and BARD1 are requires in the negative regulation of apoptotic process 43,81 . Altogether, a strong overlap of ERBB2-driven pathways was found with our homologous recombination factors, which may help define a signature to select bladder cancer patients who may benefit from targeted therapy and may use to evaluate drug response for patients.

Conclusions
To our knowledge, this is the first time where the relationship between RAD21, RAD50, BARD1 and ERBB2 was highlighted in bladder cancer. This study provided novel findings and potential prognostic markers in this type of cancer. Importantly, here we showed that high RAD21, RAD50 or BARD1 mRNA expression in bladder cancer patients with low-ERBB2 exhibit poor survival. In addition, gene expression of BARD1 alone or in combination with RAD50 acted as an independent prognostic factor for worst survival. We also identified several promising candidate genes between our targets which could be incorporated in tumor prognosis. The fact that this is a retrospective observational study is the main limitation of our work, therefore further analysis is needed. Additionally, we recognize that the median value method we used to divide the dataset into two groups based on expression levels may also have limitations due to the small sample size and limited clinical data available. In future studies, we plan to utilize more advanced methods that can accommodate larger sample sizes and more comprehensive clinical data. This is to better assess the clinical relevance of differentially expressed genes and identify potential biomarkers for bladder cancer prognosis. Also, the exact molecular mechanism between our homologous recombination targets and ERBB2 still need to be investigated to improve prognosis and treatment efficacy in bladder cancer. Using bioinformatical analysis tools to find potential overlapping gene is a good step, though validating these finding with experimental test is a must to understand the mechanism.